Control of Stochastic Gene Expression by Host Factors at the HIV Promoter

The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-κB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-κB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-κB sites exhibit distinct properties, with κB site I serving a stronger activating role than κB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-κB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally “dampen” transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency

High-Temperature Electronic Materials: Silicon Carbide and Diamond

The physical and chemical properties of wide-band-gap semiconductors make these materials an ideal wide bandgapsemiconductor choice for device fabrication for applications in many different areas, e.g. light emitters, high-temperature and high-power electronics, high-power microwave devices, micro-electromechanical system (MEM) technology, and substrates for semiconductor preparation. These semiconductors have micro-electromechanical system (MEMS) been recognized for several decades as being suitable for these applications, but until recently the low material quality has not allowed the fabrication of high-quality devices. In this material quality chapter, we review the wide-band-gap semiconductors, silicon carbide and diamond. Silicon carbide electronics is advancing from the research stage to commercial production. The commercial availability of single-crystal SiC substrates during the early 1990s gave rise to intense activity in the development of silicon carbide devices. The commercialization started with the release of blue light-emitting diode (LED). The recent release of high-power Schottky diodes was a further demonstration of the progress made towards defect-free SiC substrates. Diamond has superior physical and chemical properties. Silicon-carbide- and diamond-based diamondsilicon carbide (SiC) electronics are at different stages of development. The preparation of high-quality single-crystal substrates of wafer size has allowed recent significant progress in the fabrication of several types of devices, and the development has reached many important milestones. However, high-temperature studies are still scarce, and diamond-based electronics is still in its infancy